Trypanosomatid parasites cause devastating and life-threatening human diseases including African Sleeping Sickness, Chagas disease and Leishmaniasis. Current drug treatments are inadequate, often toxic, and no vaccines are available. With several biological properties being pursued as potential drug targets for much needed parasite-selective chemotherapeutic interventions, nuclear DNA replication has been historically overlooked presumably because it was assumed that the replication machinery was going to be conserved among eukaryotes. Post-genome sequencing, we now know that several aspects of DNA replication initiation and regulation are divergent or completely lacking in trypanosomatids, and that there are fewer replication origins than in other model eukaryotes. However, the machinery for replication fork progression, and how fork progression is regulated in the context of large polycistronic transcription units is poorly understood. There are many layers of regulation governing DNA replication to ensure that genetic information is accurately transmitted to progeny cells, and the recent links to antigenic variation in Trypanosoma brucei suggest additional layers of regulation are likely. The identification of factors necessary that promote replication fork progression and stability is likely to uncover fundamental processes necessary to maintain genome stability, and trypanosome-specific factors that might serve as selective drug targets. Here we propose to adapt an innovative methodology called iPOND (isolation of proteins on nascent DNA) to study DNA replication in T. brucei, an extracellular pathogen with well-established genetic tools. Aim 1 of this proposal will identify a comprehensive list of the DNA replication machinery (and associated regulatory factors) by coupling iPOND with mass spectrometry. Additionally, we will acquire temporal information about chromatin deposition and maturation by comparing protein profiles obtained under brief label conditions (pulse) vs. pulse-chase conditions. Aim 2 will assess changes in the protein profile under replication stress conditions to identify the trypanosomatid replication restart machinery. Together these studies will provide crucial information about trypanosome-specific factors required for DNA replication, a process conserved in all trypanosomatids, and interesting new insights into the diverse mechanisms by which eukaryotes accomplish a fundamental mechanism such as DNA synthesis. Additionally, successful completion of this proposed work will generate new insights into proteins that function at the crossroads of transcription, chromatin remodeling and DNA replication.